US20070155000A1 - Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities - Google Patents

Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities Download PDF

Info

Publication number
US20070155000A1
US20070155000A1 US11/560,490 US56049006A US2007155000A1 US 20070155000 A1 US20070155000 A1 US 20070155000A1 US 56049006 A US56049006 A US 56049006A US 2007155000 A1 US2007155000 A1 US 2007155000A1
Authority
US
United States
Prior art keywords
strain
batch
adh6
ethanol
hmf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/560,490
Other languages
English (en)
Inventor
Anneli Nilsson
Gunnar Lidén
Marie-Francoise Gorwa-Grauslund
Bärbel Hahn-Hägerdal
Carl Modig
Joao Moreira De Almeida
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scandinavian Technology Group AB
Original Assignee
Forskarpatent I SYD AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forskarpatent I SYD AB filed Critical Forskarpatent I SYD AB
Assigned to FORSKARPATENT I SYD AB reassignment FORSKARPATENT I SYD AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NILSSON, ANNELI, HAHN-HAGRDAL, BARBEL, MOREIRA DE ALMEIDA, JOAO RICARDO, GORWA-GRAUSLUND, MARIE-FRANCOISE, LIDEN, GUNNAR, MODIG, CARL TOBIAS
Publication of US20070155000A1 publication Critical patent/US20070155000A1/en
Priority to US12/201,292 priority Critical patent/US20090061502A1/en
Assigned to SCANDINAVIAN TECHNOLOGY GROUP AB reassignment SCANDINAVIAN TECHNOLOGY GROUP AB CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: FORSKARPATENT I SYD AB
Priority to US13/545,301 priority patent/US20130023021A1/en
Priority to US13/545,343 priority patent/US20130023022A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a Saccharomyces cerevisiae strain being able to grow and produce ethanol in the presence of inhibiting compounds and substances, in particular furfural and derivatives thereof while reducing such compounds, and in particular it relates to a strain that is able to produce ethanol in a fed-batch or continuous production system.
  • ethanol produced from renewable resources is considered an attractive alternative for partly replacing fossil fuels (1).
  • Many sources of lignocellulosic materials e.g. wood, forest residues and agricultural residues
  • lignocellulosic materials can potentially be used for ethanol production (2).
  • the cellulose and the hemicellulose in the lignocellulose Prior to fermentation, however, the cellulose and the hemicellulose in the lignocellulose must be converted to monomeric sugars by a combination of physical (e.g. grinding, steam explosion), chemical (e.g. dilute acid) and perhaps also enzymatic treatments (2).
  • monomeric sugars In addition to monomeric sugars also a number of other compounds are formed during these processes, several of which are potent inhibitors.
  • microorganism used for fermentation of hydrolyzates should consequently exhibit three characteristics: a) it should have high ethanol tolerance, b) it should be resistant to inhibitors found in the hydrolyzate and c) it should have a broad substrate utilization range, since the hydrolyzate contains several different sugars.
  • the quantitively most important sugars in hydrolyzate from spruce are glucose, mannose and xylose (6).
  • Saccharomyces cerevisiae is the preferred microorganism for conversion of hydrolyzate to ethanol. It has also been shown that this yeast species is more tolerant to inhibitors such as acetic acid, furfural and 5-hydroxy-methyl furfural (HMF) than several other potential production microorganisms (8). The tolerance to, in particular, many of the aldehyde compounds can most likely be explained by a bioconversion of these compounds by the yeast to, in general, the less inhibitory corresponding alcohols. It is for instance known that S. cerevisiae converts furfural into the less inhibiting compound furfuryl alcohol (9, 10). With respect to sugar utilization, S.
  • HMF 5-hydroxy-methyl furfural
  • yeast species e.g. Pichia stipitis and Candida shehatae are able to convert xylose into ethanol.
  • these yeasts have a relatively low ethanol and inhibitor tolerance, and, furthermore, require microaerobic conditions in order to give a high productivity (8, 11).
  • xylose is channeled into the pentose phosphate pathway (PPP) in a three-step process.
  • PPP pentose phosphate pathway
  • Xylose is first converted to xylitol by a xylose reductase (XR).
  • Xylitol is then oxidized to xylulose by xylitol dehydrogenase (XDH), and finally, xylulose is phosphorylated to xylulose 5-phosphate by xylulose kinase (XK) (12).
  • the first two enzymes are lacking in S. cerevisiae .
  • the activity of XK in S. cerevisiae has been shown to be low (13), which has been suggested to limit the consumption rate in S. cerevisiae strains expressing XR and XDH (14).
  • strain background appears to be important for the effect of XK (15, 16).
  • TMB3006 a genetically modified xylose-utilizing strain of S. cerevisiae was studied: TMB3006 (17). This strain express the heterologous genes XYL1 and XYL2 (encoding the enzymes XR and XDH, respectively) from P. stipitis , and overexpress the native gene XKS1 (encoding XK).
  • xylose is believed to be transported by the same uptake system as glucose, however, with a much lower affinity (23). It is also known that a concomitant uptake of glucose increases the xylose consumption rate (24). One may therefore anticipate that a higher specific conversion rate xylose in hydrolyzates can be obtained in fed-batch cultivations.
  • ADHVI may give the yeast the opportunity to survive in ligninolytic environments where products derived from lignin biodegradation may be available.
  • HMF being a carbohydrate derived product.
  • the paper does not discuss, or experimentally investigate, the potential role of ADHVI in protection against or conversion of inhibitors resulting from breakdown of the carbohydrates such as cellulose and/or hemicellulose.
  • the four strains of Saccharomyces cerevisiae used are given in Table 1.
  • the strains were maintained on agar plates with the following composition: 10 g/l yeast extract, 20 g/l soy peptone, 20 g/l agar and 20 g/l glucose.
  • Inoculum cultures were grown in 300 ml cotton plugged E-flasks with 100 ml of synthetic media according to Taherzadeh et. al. (25) with 15 g/l glucose as carbon and energy source.
  • the inoculum cultures were grown for 24 h at 30° C. and with a shaker speed of 150 rpm before 20 ml was added to the fermentor to start the cultivation.
  • the hydrolyzate used was produced from forest residue, originating mainly from spruce, in a two-stage dilute-acid hydrolysis process using sulphuric acid as the catalyst (19).
  • the hydrolyzates obtained from the two stages were mixed and stored at 8° C. until used.
  • the composition of the hydrolyzate is given in Table 2.
  • the second type of experiment was a fed-batch experiment, in which the hydrolyzate feed rate was controlled using a step-response method developed by Nilsson et. al. (19). In short, the feed-rate was changed in a step-wise manner, in which the step increase was proportional to the derivative of the measured carbon dioxide evolution rate from the previous step. Feed rate control was obtained by controlling the frequency of a peristaltic pump (Watson-Marlow Alitea AB, Sweden). Also in these experiments, a total of 1.5 l of hydrolyzate was added.
  • a gas monitor (model 1311, Brüel and Kjaer, Denmark) (described by Christensen et.al.) was used to measure the carbon dioxide evolution rate (CER).
  • the gas analyzer had three channels for measurement of carbon dioxide, oxygen and ethanol in the off-gas from the reactor.
  • the ethanol signal was calibrated against ethanol concentrations measured in the broth by HPLC, since it was assumed that the ethanol in the gas phase was in equilibrium with the ethanol in the broth. Calibration for oxygen and carbon dioxide was done using a gas containing 20.0% oxygen and 5.0% carbon dioxide.
  • a flow-injection-analysis (FIA) system (26) was used to measure biomass concentration in the reactor. This was done by measuring the optical density at 610 nm, at a frequency of 1 h ⁇ 1 . After every fermentation the FIA-signal was calibrated against measured dry-weight. Duplicate 10 ml samples of the fermentation broth were centrifuged at 3000 rpm for 3 min in pre-weighted tubes. The cells were washed with distilled water, centrifuged again and dried over night at 105° C. before they were weighted again. The dry weight was measured three times during each fermentation.
  • FIA flow-injection-analysis
  • CFU colony forming units
  • Samples for analysis of metabolite concentrations were taken regularly from the reactor. The samples were centrifuged and filtered trough 0.2 ⁇ m filters. The concentrations of glucose, mannose, xylose, galactose and arabinose were measured on an Aminex HPX-87P column (Bio-Rad, USA) at 80° C. The concentrations of ethanol, HMF, furfural, glycerol and acetic acid were measured on an Aminex HPX-87H column (Bio-Rad, USA) at 65° C. All compounds were detected with a refractive index detector, except for HMF and furfural, which were detected with a UV-detector (210 nm).
  • the mole fraction of ethanol in the gas phase was assumed to be proportional to the mole fraction of ethanol in the liquid phase.
  • the amount of evaporated ethanol could thereby be estimated by integration of the gas flow leaving the reaction multiplied with the mole fraction of ethanol in the gas, as described by Nilsson et.al., (18).
  • Cell extracts were prepared for measurements of enzyme activities in the strains TMB3000 and CBS 8066. Crude extracts were made using Y-PER reagent (Pierce, Rockford, Ill., USA). The cell extracts were kept in an ultra freezer ( ⁇ 80° C.) until used. The protein content in the cell free preparation was determined by Coomassie Protein Assay Reagent using bovine serum albumin as a standard (Pierce, Rockford, Ill., USA).
  • Furfural and HMF reducing activity was measured according to Wahlbom et. al. (27). 20 ⁇ l of the cell free extract (diluted ten times in 100 mM phosphate buffer) was diluted in 2.0 ml of 100 mM phosphate buffer (50 mM KH 2 PO 4 and 50 mM K 2 HPO 4 ) and furfural was added to a concentration of 10 mM. The samples were heated to 30° C. and thereafter the reaction was started by addition of NADH to a concentration of 100 ⁇ M. The oxidation of NADH was followed as the change in absorbance at 340 nm. The same procedure was repeated with NADPH as the co-factor, but the sample amount was increased to 200 ⁇ l due to the lower activity. The total volume was still 2.0 ml and the concentrations of furfural and NADH 10 mM and 100 ⁇ M respectively.
  • HMF reduction capacity 200 ⁇ l of diluted cell extract was used except for the measurement for strain TMB3000 with NADH as the co-factor where 20 ⁇ l sample was used due to the higher activity.
  • concentration of HMF was 10 mM. Activities were measured with both NADH and NADPH.
  • ADH activity was measured according to Bruinenberg et. al. (28).
  • the cell free extract was diluted 10 times and 20 ⁇ l of this dilution was added to 2.0 ml of 100 mM phosphate buffer. Ethanol was added to give a concentration of 100 mM. After heating to 30° the reaction was started by addition of NAD+ to a concentration of 100 ⁇ M. The reduction of NAD+ was followed as the change in absorbance at 340 nm.
  • cell samples for mRNA analysis were taken both after feeding the reactor with media without HMF and with media including 0.5 g HMF l ⁇ 1 . To get a steady state in the reactor the samples were taken 5 resident times after start of feed or change in feed media.
  • RNA samples from the reactor were spinned in ice at 3000 rpm for 1 min and thereafter frozen in liquid nitrogen and stored at ⁇ 80 C until mRNA was isolated from the samples.
  • the mRNA was isolated using Fast RNA kit (Q-biogene, USA).
  • the mRNA was then purified, cDNA synthesized, in-vitro transcribed and fragmented as described by Affymetrix. Hybridization, washing, staining and scanning of microarray-chips (Yeast Genome S98 Arrays) were made with a Affymetrix Gene Chip Oven 640, a Fluidics Station 400 and a GeneArray Scanner (Affymetrix).
  • Selected strains (over expressing LAT1, ALD6, ADH5, ADH6, GDH3, OYE3, IDP3, ADH7, AAD15, ERG27, HMG1, LYS5, SPS19, SGE1) from the ExClone collection (Resgen, Invitrogen Corporation (UK)) were grown in 300 ml shake flasks (with carbon dioxide traps) containing 100 ml SD-Ura omission media and 40 g/l glucose as described by the supplier. However, 80 ⁇ M of Cu 2+ was added when the shake flasks were inoculated and a 100 mM phosphate buffer were used. Samples for enzyme activity measurements were taken after 16 hours of growth at 30° C. and 150 rpm.
  • strain CBS 8066 The specific ethanol productivity obtained with the strain CBS 8066 was clearly lower than for the other strains, and it gradually decreased throughout the fermentation for this strain, although the hexose sugars glucose and mannose were eventually completely consumed by all strains. None of the strains were able to grow in batch culture on hydrolyzate, but there were large differences with respect to maintenance of viability. The viability of strain CBS 8066 decreased to 16% within a few hours after the start of hydrolyzate fermentation (Table 2).
  • the productivity, viability and conversion of HMF the commercial baker's yeast was somewhere in between those of CBS 8066 and TMB3000.
  • Xylose was assumed to be converted to ethanol, since no xylitol was detected ( FIG. 3 ).
  • the feed-rate was set to 25 ml/h 17 hours after the start of the experiment and after 31 hours the feed-rate was decreased to 12.5 ml/h ( FIG. 3 ).
  • the xylose consumption for TMB3006 increased to 62%. However, 55% of the xylose consumed by TMB3006 was converted to xylitol.
  • Furfural and HMF reduction capacity was measured on cell extract sampled during fed-batch experiments.
  • the enzyme activities were measured with both NADH and NADPH as co-factors ( FIG. 4 ).
  • the activities were higher for TMB3000 than for CBS 8066 in all cases.
  • the largest difference was seen for HMF reduction activity using NADH as co-factor. This activity was very low in CBS 8066, but several hundredfold higher in TMB3000. Also with NADPH as co-factor it was higher, but only about 4 times higher.
  • the ADH activity was on average 40% higher than for CBS 8066 ( FIG. 6 ).
  • the difference in furfural conversion can thus not be explained by the mere difference in total ADH activity, but may be related to differences in the relative activity of different forms of ADH or strain specific changes in the ADH protein.
  • furfural-containing chemostat cultivation both anaerobic and aerobic
  • the determining rate is the rate of reduction to furfuryl alcohol
  • the critical rate is instead the oxidation rate to form furoic acid.
  • the furfural concentration increases in the medium, which presumably leads to inhibition of a number of key enzymes, including PDH and AIDH and washout occurs.
  • the furfural concentration may 1 g/l up to 3 g/l depending on its origin.
  • furfuryl alcohol Normally the furfuryl alcohol will be measured as the fermentation is anaerobic, and the product is then almost exclusively furfuryl alcohol.
  • the transformation capacity, conversion rate determines how fast it is possible to add the furans. If they should be added too fast, furans will be accumulated in the medium, which will lead to an inhibition of central functions by means of interactions between furans and a number of enzymes such as PDH, PDC and others. This in turn leads to an inhibitor growth and down-regulation of the fermentation rate.
  • the average enzyme activities for furfural and HMF conversion in CBS 8066 was similar to those found for strain TMB3001, a strain derived from CEN.PK PK113-7A.
  • the average activities for furfural conversion in CBS 8066 were 353 mU/mg protein with NADH as co-factor and 22.8 mU/mg protein with NADPH as co-factor, compared to 490 and 22 mU/mg protein, respectively, found in TMB3001.
  • the average activities for CBS 8066 were 1.8 mU/mg protein (NADH) and 12.4 mU/mg protein (NADPH), compared with 2.2 and 22 mU/mg protein, respectively, for TMB3001.
  • the Enzyme activities obtained for the strain TMB3000 were very different.
  • the average furfural reduction activity was several times higher than for CBS 8066 and TMB3001, although the co-factor preference was similar.
  • the most striking difference was, however, the high activity for HMF reduction with NADH as co-factor ( FIG. 4 ).
  • This activity was in fact more than 150 times higher for TMB3000 than for the other two strains.
  • the NADPH coupled reduction rate was several times higher for TMB3000 than for CBS 8066.
  • the furfural and HMF conversion activities provide an explanation for the advantage of TMB3000 over CBS 8066 in lignocellulose fermentation.
  • High activities ensure high conversion rates of furfural and HMF, and possibly other inhibitory aldehydes (32), so that the concentration of these inhibitors is kept low in the fermentation.
  • strain CBS 8066 the measured in vitro activity for furfural reduction would correspond to an in vivo reduction rate of 0.69 g/g h. This, in fact agrees well with the maximum conversion rate. reported in synthetic media for the same strain (0.6 g/g h).
  • the corresponding predicted specific conversion rate of HMF would be 0.03 g/g h, which is somewhat lower than the value reported in synthetic media of 0.14 g/g h.
  • strain TMB3000 measured in vitro reduction activities for furfural and HMF were 2.26 g/g h and 0.98 g/g h respectively and this would correspond to feeding rates of about 3 l/h, at the cell density and volumes used which is much higher than those applied in the present work (cf. FIG. 2 ).
  • the activities for HMF conversion correspond to a feeding rate of only 100 ml/h.
  • Another factor to consider is the difference in co-factor preference for HMF conversion. Since NADH is the preferred co-factor in the conversion of HMF, there will be no drain of NADPH competing with anabolic reactions in strain TMB3000.
  • the enzyme encoded by the gene ADH6 in Saccharomyces cerevisiae is able to convert HMF using the co-factor NADPH Yeast strains that over-express this gene have a substantially higher conversion rate of HMF in both aerobic and anaerobic cultures.
  • strains over-expressing ADH6 has a substantially higher ethanol productivity and are less effected by inhibition during fermentation of a dilute-acid lignocellulose hydrolyzate. Strains genetically modified to give a high expression of ADH6 will therefore be advantageous for the conversion of lignocellulosic hydrolyzates.
  • the alcohol dehydrogenase VI (ADH6) gene from Saccharomyces cerevisiae TMB3000 and CEN.PK 113-5D were amplified using the primers ADH6-FOR and ADH6-REV (Table 4).
  • the 5′ region of the primers ADH6-FOR and ADH6-REV contain 34 and 33 nucleotides corresponding to the sequence of the HXT promoter and PGK1 terminator, respectively.
  • the PCR products were analyzed by electrophoresis in agarose gels and purified using QIAquick PCR Purification kit (QIAGEN).
  • QIAGEN QIAquick PCR Purification kit
  • the vector pYEplacHXT was linearized using the restriction endonuclease Bam HI.
  • a mix containing the linear vector (6.2 Kb), the ADH6 product from TMB 3000 (T-ADH6) or CEN.PK 113-5D (C-ADH6) was used to transform S. cerevisiae CEN.PK 113-5D by lithium acetate method (38).
  • the cells were resuspended in 1 mL of 100 mM lithium acetate (LiAc) and incubated at 30° C. for 10-15 minutes. The suspension was centrifuged at top speed for 30 seconds, the supernatant removed and the transformation mix (240 ⁇ l PEG 50% w/v, 36 ⁇ l 1.0 M lithium acetate, 52 ⁇ l 2 mg/mL ssDNA, 28 ⁇ l sterile water, 1.0 ⁇ l 40 ng/ ⁇ l pYEplacHXT vector and 3 ⁇ l 40 ng/ ⁇ l PCR product) was added to the pellet. After subsequent incubations at 30° C. for 30 min and 42° C.
  • LiAc lithium acetate
  • yeast cells were re-suspended in 150 ⁇ l of YNB and left at room temperature for approximately 2 hours. After incubation the mix containing cells was plated on YNB-plates, which were incubated at 30° C. for 3-4 days.
  • a yeast control strain was constructed by transformation with the empty pYEplacHXT vector. Transformant yeast strains were selected by colony PCR using ADH6 primers and ethanol oxidation capacity. Plasmids from two transformants (C-ADH6-2 and T-ADH6-2) were recovered, amplified in E. coli DH5 ⁇ and submitted to automatic sequencing.
  • the hydrolyzate used was produced from forest residue, originating mainly from spruce, in a two-stage dilute-acid hydrolysis process using sulphuric acid as the catalyst (19).
  • the hydrolyzates obtained from the two stages were mixed and stored at 8° C. until used.
  • the composition of the hydrolyzate is given in Table 5.
  • Cell extracts of strains over-expressing ADH6 were prepared for measurements of enzyme activities. Crude extracts were made using Y-PER reagent (Pierce, Rockford, Ill., USA). The protein content in the cell free preparation was determined using Micro BCA Protein Assay Kit (Pierce).
  • Enzyme activities for theoxidation of ethanol and the reduction of furfural, 5-hydroxymethyl-furfural (HMF) and dihydroxyacetone phosphate (DHAP) were measured on cell extract samples.
  • the rate of ethanol oxidation was determined by monitoring the reduction of NAD + photometrically at a wavelength of 340 nm.
  • the enzyme assay, based on (37), contained 5.0 mM NAD + and 1.7 M of ethanol in 100 mM glycine buffer at pH 9.0 in 1.0 cm path length cuvettes. The samples were incubated at 30° C. and the reaction was started by addition of ethanol. HMF and furfural reducing activities were measured according to (27).
  • ADH6 gene was PCR amplified from CEN.PK or TMB3000 genomic DNA and cloned into the yeast vector pYEplac-HXT, generating pYEplacHXT-C/ADH6 and pYEplacHXT-T/ADH6 vectors respectively.
  • the plasmids were used for the transformation of CEN.PK113-5D strain.
  • Colony PCR was used to select yeast strains that carried a pYEplacHXT-ADH6 vector.
  • Clones with increased expression of ADH6 gene were selected amongst transformants for their increased ethanol oxidation capacity compared to the control strain CEN.PK113-5D carrying the empty vector YEplac-HXT ( FIG. 8 ).
  • HMF and furfural conversion capacity of ADH6 over-expressing strains was analyzed using NADH and NADPH as cofactors in enzymatic assays ( FIG. 9 ).
  • Strains overexpressing ADH6 were able to convert HMF using NADPH as well as NADH as cofactor, but the activity using NADH was clearly lower than with NADPH.
  • similar values for HMF and furfural conversion were obtained for C- and T-ADH6 strains, which suggest no differences in protein structures/activity between CEN.PK and TMB3000 strains. Indeed, the ADH6 gene sequences from the two strains did not show any significant difference, except for a substitution of the G-203 in C-ADH6-2 for E-203 in T-ADH6-2.
  • the control strain and a strain over-expressing ADH6 from TMB3000 were used in anaerobic batch fermentations with a dilute-acid hydrolyzate ( FIG. 11 and 12 ).
  • T/ADH6-2 strain was clearly less inhibited than the control strain and the CER did not decrease as rapidly for T/ADH6-2 as for the control strain ( FIG. 11 and 12 ). This is also reflected in the specific ethanol productivity, which was 35% higher for T/ADH6-2 compared to the control strain.
  • the specific uptake rate of HMF was found to be five-fold higher in the T/ADH6-2 than in the control strain (0.05 g/g h and 0.01 g/g h, for T/AHD6-2 and the control strain, respectively).
  • the specific uptake rate of furfural was, however, the same (0.02 g/g h) for both strains, showing that the tolerance is not linked to the furfural, but to the HMF conversion capacity.
  • a strain overexpressing ADH6 had a 35% higher fermentation rate of undetoxified dilute-acid hydrolyzate than the corresponding control strain.
  • TABLE 1 Description of the five different strains of S. cerevisae used in this work. Strain Description Reference CBS 8066 A widely used diploid laboratory strain (21) Baker's Commercially available yeast obtained from — yeast the Swedish Baker's yeast company, Jabelbolaget AB, Rotebro, Sweden TMB3000 A strain isolated from a spent sulfite (32) liquor fermentation plant TMB3006 A genetically modified strain based on (17) MB3000. Expresses the heterologous genes XYL1 and XYL2 from P. stipitis and overexpresses the gene XKS1 from S. cerevisiae .
  • FIG. 1 After an initial batch on synthetic media, 1.5 liters of hydrolyzate was added to the reactor. Top row: Batch fermentations where hydrolyzate was added with maximal rate (approximately 2000 ml/h). Bottom row: Fed-batch where the feed-rate was controlled by the program previously developed (see Materials and Methods). Both batch and fed-batch fermentations was performed with A: CBS 8066, B: Baker's yeast, C: TMB3000, D: TMB3006. Left scale: carbon evolution rate (CER). Right scale: ethanol and feed-rate. The amount of formed biomass and the concentrations of HMF and furfural can be seen in FIG. 2 .
  • FIG. 2 Batch and fed-batch fermentations with A: CBS 8066, B: Baker's yeast, C: TMB3000, D: TMB3006.
  • A CBS 8066
  • B Baker's yeast
  • C TMB3000
  • D TMB3006.
  • the CER, feed-rate and amount of formed ethanol from these experiments can bee seen in FIG. 1 .
  • Top row Batch fermentations where hydrolyzate was added with maximal rate (approximately 2000 ml/h).
  • Bottom row Fed-batch where the feed-rate was controlled by the program previously developed (see Materials and Methods).
  • Left scale biomass.
  • Right scale HMF and furfural concentrations.
  • FIG. 3 Fermentations with TMB3006.
  • A Batch fermentation where 1.5 liter of hydrolyzate with maximum feeding rate of approximately 2000 ml/h.
  • B Fed-batch with the previously developed control strategy.
  • C Fed-batch fermentation with a low feed-rate. 17 hours after the start of the initial batch the feed-rate is set to 25 ml/h. At 48 hours the feed-rate is decreased to 12.5 ml/h until totally 850 ml of hydrolyzate has been added.
  • Right scale xylose and xylitol concentration.
  • Left scale xylose consumption.
  • FIG. 4 Enzyme activity measurements from fed-batches with CBS 8066 (white bars) and TMB3000 (gray bars).
  • A Enzyme activity for conversion of furfural.
  • B Enzyme activity for the conversion of HMF.
  • Top row NADH used as the co-factor.
  • Bottom row NADPH used as the co-factor.
  • FIG. 6 Array expression for different genes. Black bars: mRNA from TMB3000, Striped bars: mRNA from TMB3000 grown on synthetic media supplemented with 0.5 g/l HMF, Grey bars: mRNA from CBS8066, White bars: mRNA from CBS8066 grown on synthetic media supplemented with 0.5 g/l HMF
  • FIG. 7 Enzymatic conversion rates of cell free extracts from the Exclone collection over expressing different genes. Black bars: conversion rate of furfural with NADH, Striped bars: conversion rate of furfural with NADPH, Grey bars: conversion rate of HMF with NADH, White bars: conversion rate of HMF with NADPH.
  • FIG. 8 Specific ethanol oxidation activity (in mU/mg protein) from cell extracts using NAD + as cofactor.
  • 113-5D CEN.PK 113-5D with empty vector YEplac-HXT
  • C/ADH6-m clone m with ADH6 gene from CEN.PK strain overexpressed
  • T/ADH6-n clone n with ADH6 gene from TMB3000 strain overexpressed.
  • FIG. 9 Specific enzyme activities in crude cell extracts for the control strain CEN.PK113-5D (YEplac-HXT) and the ADH6-overexpressing strains C/ADH-2 and T/ADH6.2.
  • A Conversion of furfural with NADH as co-factor.
  • B Conversion of HMF with NADH as the co-factor.
  • C Conversion of furfural with NADPH as the co-factor.
  • D Conversion of HMF with NADPH as the co-factor.
  • FIG. 10 Specific DHAP reduction activity in crude cell extracts for the CEN.PK113-5D (YEplacHXT) control strain, the ADH6-overexpression strains C/ADH6-2 and T/ADH6-2 and strain TMB3000, using NADH (A) and NADPH (B) as co-factor.
  • FIG. 11 Batch fermentation of a dilute-acid hydrolyzate with the control strain CEN.PK113-5D (YEplacHXT). An arrow indicates the addition of hydrolyzate.
  • FIG. 12 Batch fermentation of a dilute-acid hydrolyzate with strain T/ADH6-2. The arrow indicates the addition of hydrolyzate.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US11/560,490 2004-05-19 2006-11-16 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities Abandoned US20070155000A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US12/201,292 US20090061502A1 (en) 2004-05-19 2008-08-29 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,301 US20130023021A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,343 US20130023022A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE0401303A SE0401303D0 (sv) 2004-05-19 2004-05-19 Ethanol productivities of microbial strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
SE0401303-3 2004-05-19
PCT/SE2005/000738 WO2005111214A1 (en) 2004-05-19 2005-05-19 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2005/000738 Continuation WO2005111214A1 (en) 2004-05-19 2005-05-19 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/201,292 Division US20090061502A1 (en) 2004-05-19 2008-08-29 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,301 Continuation US20130023021A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities

Publications (1)

Publication Number Publication Date
US20070155000A1 true US20070155000A1 (en) 2007-07-05

Family

ID=32589766

Family Applications (4)

Application Number Title Priority Date Filing Date
US11/560,490 Abandoned US20070155000A1 (en) 2004-05-19 2006-11-16 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US12/201,292 Abandoned US20090061502A1 (en) 2004-05-19 2008-08-29 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,301 Abandoned US20130023021A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,343 Abandoned US20130023022A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities

Family Applications After (3)

Application Number Title Priority Date Filing Date
US12/201,292 Abandoned US20090061502A1 (en) 2004-05-19 2008-08-29 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,301 Abandoned US20130023021A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US13/545,343 Abandoned US20130023022A1 (en) 2004-05-19 2012-07-10 Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities

Country Status (10)

Country Link
US (4) US20070155000A1 (de)
EP (1) EP1766028B1 (de)
AT (1) ATE500334T1 (de)
CA (1) CA2567109C (de)
DE (1) DE602005026657D1 (de)
DK (1) DK1766028T3 (de)
ES (1) ES2361419T3 (de)
SE (1) SE0401303D0 (de)
WO (1) WO2005111214A1 (de)
ZA (1) ZA200609490B (de)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080090283A1 (en) * 2006-10-13 2008-04-17 Rowan Universtity Ethanol resistant and furfural resistant strains of E. coli FBR5 for production of ethanol from cellulosic biomass
WO2009006135A2 (en) * 2007-06-28 2009-01-08 The United States Of America, As Represented By The Secretary Of Agriculture Inhibitor tolerant saccharomyces cerevisiae strain
WO2009043012A1 (en) * 2007-09-27 2009-04-02 Mascoma Corporation Progressive fermentation of lignocellulosis biomass
WO2009094631A2 (en) * 2008-01-25 2009-07-30 Newagriculture, Inc. A novel composition of matter and method for stimulating the growth of beneficial microorganisms
US20100279361A1 (en) * 2007-05-02 2010-11-04 Mascoma Corporation Two-stage method for pretreatment of lignocellulosic biomass
US20110151532A1 (en) * 2008-05-23 2011-06-23 United of Georgia Research Foundation, Inc. Paenibacillus spp. and methods for fermentation of lignocellulosic materials
US20120138546A1 (en) * 2009-07-13 2012-06-07 Andrea Bonanni High Temperature Lignin Separation Process
US20120196342A1 (en) * 2011-02-01 2012-08-02 Rachel Ruizhen Chen Industrial Applications of A Novel Aldo/Keto Reductase Of Zymomonas Mobilis
US8669076B1 (en) 2013-03-11 2014-03-11 E I Du Pont De Nemours And Company Cow rumen xylose isomerases active in yeast cells
WO2014098939A1 (en) 2012-12-20 2014-06-26 E. I. Du Pont De Nemours And Company Expression of xylose isomerase activity in yeast
WO2014164410A1 (en) 2013-03-11 2014-10-09 E. I. Du Pont De Nemours And Company Bacterial xylose isomerases active in yeast cells
US20160090405A1 (en) * 2014-05-20 2016-03-31 Lawrence Livermore National Security, Llc Engineered Microorganisms Having Resistance to Ionic Liquids
WO2017176875A1 (en) 2016-04-08 2017-10-12 E I Du Pont De Nemours And Company Arabinose isomerases for yeast
US9951359B2 (en) 2013-06-25 2018-04-24 Ut-Battelle, Llc Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification
CN110317798A (zh) * 2019-04-15 2019-10-11 中国科学技术大学 一种醇脱氢酶及其制备醇的应用

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150328347A1 (en) 2005-03-24 2015-11-19 Xyleco, Inc. Fibrous materials and composites
US7708214B2 (en) 2005-08-24 2010-05-04 Xyleco, Inc. Fibrous materials and composites
WO2008133665A2 (en) * 2006-12-07 2008-11-06 Massachusetts Institute Of Technology Global transcription machinery engineering
WO2009017441A1 (en) * 2007-07-31 2009-02-05 Bärbel Hahn Ab Polypeptide having nadh dependent hmf reductase activity
WO2009137804A1 (en) * 2008-05-09 2009-11-12 University Of Georgia Research Foundation, Inc. Yeast cells and mehtods for increasing ethanol production
ES2672348T3 (es) 2008-12-24 2018-06-14 Dsm Ip Assets B.V. Genes de la isomerasa de xilosa y su uso en la fermentación de azúcares pentosas
CN102947440B (zh) 2009-12-30 2015-04-08 埃欧金能源公司 呈现增强的木质纤维素水解产物发酵的改良酵母菌株
KR101690289B1 (ko) * 2010-03-22 2016-12-28 삼성전자주식회사 알코올 발효저해물에 대한 저항성을 증대시키는 단리된 폴리뉴클레오티드, 이를 포함하는 재조합 벡터, 알코올 생산성 형질전환 균주 및 이를 이용한 알코올의 제조방법
CA2817707C (en) 2010-11-15 2020-05-05 Scandinavian Technology Group Ab New strains of saccharomyces cerevisiae with improved ethanol production, improved xylose conversion and reduced xylitol production
DK2663645T3 (da) 2010-11-18 2015-03-23 Dsm Ip Assets Bv Gærstammer, der er modificeret til produktion af ethanol fra glycerol
WO2012125027A1 (en) 2011-03-14 2012-09-20 Dsm Ip Assets B.V. Yeast strains that ferment uronic acids
EP2546336A1 (de) 2011-07-11 2013-01-16 DSM IP Assets B.V. Hefestränge, die Uronsäure verbrauchen und Fermentierungsprodukte wie Ethanol herstellen
CN103073633A (zh) * 2011-10-25 2013-05-01 天津科技大学 一种抗5-羟甲基糠醛的抗体
EP2785849B1 (de) 2011-11-30 2017-09-27 DSM IP Assets B.V. Manipulierte hefestämme zur herstellung von ethanol aus acetatsäure und glycerol
WO2014033019A1 (en) 2012-08-28 2014-03-06 Dsm Ip Assets B.V. Yeast strains engineered to produce ethanol from acetate
WO2014033018A1 (en) 2012-08-28 2014-03-06 Dsm Ip Assets B.V. Yeast strains engineered to produce ethanol from acetate
WO2014086702A2 (de) * 2012-12-03 2014-06-12 Basf Se Enzymatische reduktion von hydroxymethylfurfuralen
AR097480A1 (es) 2013-08-29 2016-03-16 Dsm Ip Assets Bv Células de levadura convertidoras de glicerol y ácido acético con una conversión de ácido acético mejorada
CN103627644B (zh) * 2013-10-26 2015-09-30 三峡大学 一种酿酒酵母变异菌株及其应用
KR20150065213A (ko) 2013-12-04 2015-06-15 삼성전자주식회사 감소된 에탄올 생산능을 갖는 효모 세포 및 이의 용도
US20180030482A1 (en) 2014-04-18 2018-02-01 Moralco B.V. Use of acetaldehyde in the fermentative production of ethanol
CN105462867B (zh) * 2015-11-27 2019-07-16 华南理工大学 一株耐受高浓度糠醛的酿酒酵母及其应用
CN112813129B (zh) * 2021-02-05 2023-09-08 江南大学 利用区室化提高酵母菌中7-脱氢胆固醇产量的方法

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053793A1 (en) * 2006-10-13 2009-02-26 Rowan University Ethanol resistant and furfural resistant strains of E. coli FBR5 for production of ethanol from cellulosic biomass
US20090061501A1 (en) * 2006-10-13 2009-03-05 Rowan University Ethanol resistant and furfural resistant strains of E. coli FBR5 for production of ethanol from cellulosic biomass
US20080090283A1 (en) * 2006-10-13 2008-04-17 Rowan Universtity Ethanol resistant and furfural resistant strains of E. coli FBR5 for production of ethanol from cellulosic biomass
US20100279361A1 (en) * 2007-05-02 2010-11-04 Mascoma Corporation Two-stage method for pretreatment of lignocellulosic biomass
WO2009006135A2 (en) * 2007-06-28 2009-01-08 The United States Of America, As Represented By The Secretary Of Agriculture Inhibitor tolerant saccharomyces cerevisiae strain
WO2009006135A3 (en) * 2007-06-28 2009-02-26 Us Agriculture Inhibitor tolerant saccharomyces cerevisiae strain
WO2009043012A1 (en) * 2007-09-27 2009-04-02 Mascoma Corporation Progressive fermentation of lignocellulosis biomass
US20110081697A1 (en) * 2007-09-27 2011-04-07 Chaogang Liu Progressive Fermentation of Lignocellulosic Biomass
WO2009094631A2 (en) * 2008-01-25 2009-07-30 Newagriculture, Inc. A novel composition of matter and method for stimulating the growth of beneficial microorganisms
US20110053224A1 (en) * 2008-01-25 2011-03-03 Yangming Martin Lo Novel composition of matter and method for stimulating the growth of beneficial microorganisms
WO2009094631A3 (en) * 2008-01-25 2009-10-22 Newagriculture, Inc. A novel composition of matter and method for stimulating the growth of beneficial microorganisms
US9458422B2 (en) 2008-01-25 2016-10-04 University Of Maryland Composition of matter and method for stimulating the growth of beneficial microorganisms
US20110151532A1 (en) * 2008-05-23 2011-06-23 United of Georgia Research Foundation, Inc. Paenibacillus spp. and methods for fermentation of lignocellulosic materials
US8652819B2 (en) * 2008-05-23 2014-02-18 University Of Georgia Research Foundation, Inc. Paenibacillus spp. and methods for fermentation of lignocellulosic materials
US20120138546A1 (en) * 2009-07-13 2012-06-07 Andrea Bonanni High Temperature Lignin Separation Process
US20120196342A1 (en) * 2011-02-01 2012-08-02 Rachel Ruizhen Chen Industrial Applications of A Novel Aldo/Keto Reductase Of Zymomonas Mobilis
WO2014098939A1 (en) 2012-12-20 2014-06-26 E. I. Du Pont De Nemours And Company Expression of xylose isomerase activity in yeast
US8669076B1 (en) 2013-03-11 2014-03-11 E I Du Pont De Nemours And Company Cow rumen xylose isomerases active in yeast cells
WO2014164410A1 (en) 2013-03-11 2014-10-09 E. I. Du Pont De Nemours And Company Bacterial xylose isomerases active in yeast cells
US8906656B2 (en) 2013-03-11 2014-12-09 E I Du Pont De Nemours And Company Cow rumen xylose isomerases active in yeast cells
US9187743B2 (en) 2013-03-11 2015-11-17 E I Du Pont De Nemours And Company Bacterial xylose isomerases active in yeast cells
US9371535B2 (en) 2013-03-11 2016-06-21 E I Du Pont De Nemours And Company Bacterial xylose isomerases active in yeast cells
US9951359B2 (en) 2013-06-25 2018-04-24 Ut-Battelle, Llc Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification
US20160090405A1 (en) * 2014-05-20 2016-03-31 Lawrence Livermore National Security, Llc Engineered Microorganisms Having Resistance to Ionic Liquids
US10370418B2 (en) * 2014-05-20 2019-08-06 Lawrence Livermore National Security, Llc Engineered microorganisms having resistance to ionic liquids
WO2017176875A1 (en) 2016-04-08 2017-10-12 E I Du Pont De Nemours And Company Arabinose isomerases for yeast
CN110317798A (zh) * 2019-04-15 2019-10-11 中国科学技术大学 一种醇脱氢酶及其制备醇的应用

Also Published As

Publication number Publication date
DE602005026657D1 (de) 2011-04-14
DK1766028T3 (da) 2011-05-02
US20090061502A1 (en) 2009-03-05
ATE500334T1 (de) 2011-03-15
ZA200609490B (en) 2008-07-30
US20130023022A1 (en) 2013-01-24
EP1766028B1 (de) 2011-03-02
WO2005111214A1 (en) 2005-11-24
CA2567109A1 (en) 2005-11-24
EP1766028A1 (de) 2007-03-28
CA2567109C (en) 2014-07-29
SE0401303D0 (sv) 2004-05-19
ES2361419T3 (es) 2011-06-16
US20130023021A1 (en) 2013-01-24

Similar Documents

Publication Publication Date Title
CA2567109C (en) Ethanol productivities of saccharomyces cerevisiae strains in fermentation of dilute-acid hydrolyzates depend on their furan reduction capacities
US11326175B2 (en) Fermentative glycerol-free ethanol production
Petersson et al. A 5‐hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance
Almeida et al. NADH-vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae
Lewis Liu et al. Multiple gene-mediated NAD (P) H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae
Chu et al. Genetic improvement of Saccharomyces cerevisiae for xylose fermentation
Almeida et al. Stress‐related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae
Wahlbom et al. Furfural, 5‐hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae
Toivari et al. Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability
Matsushika et al. Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives
Tomás-Pejó et al. Adaptation of the xylose fermenting yeast Saccharomyces cerevisiae F12 for improving ethanol production in different fed-batch SSF processes
Meinander et al. Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1
Träff et al. Putative xylose and arabinose reductases in Saccharomyces cerevisiae
Laadan et al. Identification of an NADH‐dependent 5‐hydroxymethylfurfural‐reducing alcohol dehydrogenase in Saccharomyces cerevisiae
Pitkänen et al. Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain
Almeida et al. Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)
Modig et al. Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate
Du et al. The production of ethanol from lignocellulosic biomass by Kluyveromyces marxianus CICC 1727-5 and Spathaspora passalidarum ATCC MYA-4345
JP2011024500A (ja) 発酵能力が向上された酵母及びその利用
Rodrigues et al. Enhanced xylitol production by precultivation of Candida guilliermondii cells in sugarcane bagasse hemicellulosic hydrolysate
Novy et al. Saccharomyces cerevisiae strain comparison in glucose–xylose fermentations on defined substrates and in high-gravity SSCF: convergence in strain performance despite differences in genetic and evolutionary engineering history
Almeida et al. Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction
CN108603179B (zh) 发酵产物生产增加的真核细胞
Dmytruk et al. Development of the thermotolerant methylotrophic yeast Hansenula polymorpha as efficient ethanol producer
Laadan et al. Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants

Legal Events

Date Code Title Description
AS Assignment

Owner name: FORSKARPATENT I SYD AB, SWEDEN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NILSSON, ANNELI;LIDEN, GUNNAR;GORWA-GRAUSLUND, MARIE-FRANCOISE;AND OTHERS;REEL/FRAME:019033/0924;SIGNING DATES FROM 20070207 TO 20070228

AS Assignment

Owner name: SCANDINAVIAN TECHNOLOGY GROUP AB, SWEDEN

Free format text: CHANGE OF NAME;ASSIGNOR:FORSKARPATENT I SYD AB;REEL/FRAME:021780/0035

Effective date: 20081009

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION